Modulation of Cas9 level for efficient CRISPR-Cas9-mediated chromosomal and plasmid gene deletion in Bacillus thuringiensis.

OBJECTIVES

To set up an efficient gene editing system in Bacillus thuringiensis (Bt) using CRISPR-Cas9 by demonstrating deletion of chromosomal and plasmid genes.

RESULTS

CRISPR-Cas9 from Streptococcus pyogenes was found to function in Bt cells, resulting in DNA cleavage that is lethal to the cells. The system was assessed for its ability to mediate gene editing by knock-out of the protease genes nprA (neutral protease A) and aprA (alkaline protease A). Gene editing was not detected when the Bacillus-derived pBCX was used to carry CRISPR-Cas9 elements and a DNA repair template. When the Cas9 promoter was replaced with the sporulation-specific promoter cyt2A, a Bt ∆nprA clone was obtained, but this plasmid construct did not give reproducible results. Bt ∆nprA ∆aprA and Bt ∆aprA deletion mutants were finally generated when the Lactobacillus plantarum-derived plasmid pLPPR9 was used, likely due to its lower copy number reducing Cas9 toxicity. Only three to four clones each needed to be screened to identify the desired gene-modified mutants. Conversely, efficient editing of the plasmid vip3A gene required the use of pBCX and longer homology sequences for the repair template.

CONCLUSIONS

Capitalizing on the differential impact of plasmid copy number and homology arm length, we devised distinct yet simple and efficient approaches to chromosomal and plasmid gene deletion for Bt that condense the screening process, minimize screening, and facilitate multiple consecutive gene editing steps.